The goals of this project are to identify intracellular mechanisms involved in the interactions among hormones that regulate Na+ and Na+-coupled organic solute absorption along the nephron, as a means of understanding the normal regulation of salt and water reabsorption. The focus of the projects will be the regulation of the amiloride-sensitive Na+ channel in the cortical (CCD) and inner medullary collecting duct (IMCD), and Na+/H+ exchange and Na+-dependent glucose transport in proximal convoluted and straight tubules. These processes will be examined using the technique of perfusing isolated segments of rat and rabbit nephron, in combination with unidirectional flux measurements, electrophysiological measurements of individual membrane conductances, and fluorescence-digital imaging microscopy (F-DIM). F-DIM will be used in conjunction with fluorescence- labeled antibodies against the amiloride-sensitive Na+ channel in order to determine its localization, mechanism of activation by arginine vasopressin (AVP), possible presence in the IMCD, modification by urine enzymes, and changes in relative density with agents that stimulate and inhibit Na+ reabsorption. The same techniques will be used with antibodies specific to the Na+-glucose cotransporter in the proximal tubule to examine its distribution along the proximal nephron and its possible regulation by membrane recycling. We will also examine the basis for the differential response of Na+ transport to AVP in the rat and rabbit CCD, as a means of understanding the regulation of luminal membrane Na+ channel activity. Of particular interest will be differential responses of rat and rabbit CCD's to: 1) Activation by prostaglandins and alpha 2-adrenergics of the inhibitory GTP-binding protein (Gi) associated with adenylate cyclase; 2) Pertussis toxin-induced ribosylation of G proteins; and 3) Activators or inhibitors of the protein kinase C and intracellular Ca++ systems. We will also examine the Na+ transport in the rat and rabbit IMCD with special regard to: 1) Characteristics of the luminal membrane Na+ entry mechanism; 2) Its response to AVP and the above modifiers of intracellular second messengers; and 3) Mechanism of inhibition of Na+ transport in the IMCD (and possibly in the CCD) by atrial natriuretic peptides, and its possible mediation through cyclic-GMP and modification of the adenylate cyclase and phosphokinase C-Ca++ regulatory systems. In the proximal tubule we will examine the mechanisms by which angiotensin II and alpha 2-adrenergic agonists increase volume absorption with regard to: 1) Regulation of Na+/H+ exchange and/or Na+ cotransport with glucose and amino acids; and 2) Intracellular mediators of the stimulatory effect.